N-type calcium channel antagonists for the treatment of pain

ABSTRACT

Compounds having selective action at neuronal N-type calcium channels useful for the treatment of pain in accord with the following structural diagram,  
                 
 
     wherein R 1 , R 2  and R 3  are a variety of groups as defined in the specification.

FIELD OF THE INVENTION

[0001] This invention relates to compounds and methods for the treatmentor prevention of pain or nociception.

RELATED ART

[0002] Pain causes a great deal of suffering and is a sensory experiencedistinct from sensations of touch, pressure, heat and cold. It is oftendescribed by sufferers by such terms as bright, dull, aching, pricking,cutting or burning and is generally considered to include both theoriginal sensation and the reaction to that sensation. This range ofsensations, as well as the variation in perception of pain by differentindividuals, renders a precise definition of pain difficult. Where painis “caused” by the stimulation of nociceptive receptors and transmittedover intact neural pathways, this is termed nociceptive pain. Pain mayalso be caused by damage to neural structures, and pain is often ismanifested as neural supersensitivity; this type of pain is referred toas neuropathic pain.

[0003] The level of stimulation at which pain is perceived is referredto as the “pain threshold”. Where the pain threshold is raised, forinstance, by the administration of an analgesic drug, a greaterintensity or more prolonged stimulus is required before pain isexperienced. Analgesics are a class of pharmaceutical agent which,following administration to a patient in need of such treatment, relievepain without loss of consciousness. This is in contrast to otherpain-relieving drugs, for example, general anaesthetics which obtundpain by producing a hiatus in consciousness, or local anaesthetics whichblock transmission in peripheral nerve fibres thereby preventing pain.

[0004] Tachykinin antagonists have been reported to induceantinociception in animals, which is believed to be analogous toanalgesia in man (for review see Maggi et al, J. Auton. Pharmacol.(1993) 13, 23-93). In particular, non-peptide NK-1 receptor antagonistshave been shown to produce such analgesia, thus, for example, inclassical tests of chemo-nociception (phenylbenzoquinone-inducedwrithing and formalin test) the NK-1 receptor antagonist RP 67,580produced analgesia with potency comparable to that of morphine (Garretet al, Proc. Natl. Acad. Sci. USA (1993) 88, 10208-10212).

[0005] Opioid analgesics are a well-established class of analgesicagents. These compounds are generally accepted to include, in a genericsense, all drugs, natural or synthetic, with morphine-like actions. Thesynthetic and semi-synthetic opioid analgesics are derivatives of fivechemical classes of compound: phenanthrenes; phenylheptylamines;phenylpiperidines; morphinans; and benzomorphans. Pharmacologicallythese compounds have diverse activities, thus some are strong agonistsat the opioid receptors (e.g. morphine); others are moderate to mildagonists (e.g. codeine); still others exhibit mixed agonist-antagonistactivity (e.g. nalbuphine); and yet others are partial agonists (e.g.nalorphine). Whilst an opioid partial agonist such as nalorphine, (theN-alkyl analogue of morphine) will antagonise the analgesic effects ofmorphine, when given alone it can be a potent analgesic in its ownright. Of all of the opioid analgesics, morphine remains the most widelyused and is a suitable archetype compound. Unfortunately, apart from itsuseful therapeutic properties, morphine also has a number of drawbacksincluding respiratory depression, decreased gastrointestinal motility(resulting in constipation) and, in some individuals, nausea andvomiting may occur. Another characteristic is the development oftolerance and physical dependence which may limit the clinical use ofsuch compounds.

[0006] Anti-inflammatory compounds directed at blocking or reducingsynovial inflammation, and thereby improving function, and analgesicsdirected to reducing pain, are presently the primary method of treatingthe rheumatoid diseases and arthritis. Aspirin and other salicylatecompounds are frequently used in treatment to interrupt amplification ofthe inflammatory process and temporarily relieve the pain. Other drugcompounds used for these purposes include phenylpropionic acidderivatives such as Ibuprofen and Naproxin, Sulindac, phenyl butazone,corticosteroids, antimalarials such as chloroquine andhydroxychloroquine sulfate, and fenemates. For a thorough review ofvarious drugs utilized in treating rheumatic diseases, reference is madeto J. Hosp. Pharm., 36:622 (May 1979).

[0007] Calcium channels are membrane-spanning, multi-subunit proteinsthat allow controlled entry of Ca⁺⁺ ions into cells from theextracellular fluid. Such channels are found throughout the animalkingdom, and have been identified in bacterial, fungal and plant cells.Commonly, calcium channels are voltage dependent. In such channels, the“opening” allows an initial influx of Ca⁺⁺ ions into the cells whichlowers the potential difference between the inside of the cell bearingthe channel and the extracellular medium bathing the cell. The rate ofinflux of Ca⁺⁺ ions into the cell depends on this potential difference.All “excitable” cells in animals, such as neurons of the central nervoussystem (“CNS”), peripheral nerve cells, and muscle cells, includingthose of skeletal muscles, cardiac muscles, and venous and arterialsmooth muscles, have voltage-dependent calcium channels. Calciumchannels are physiologically important because the channels have acentral role in regulating intracellular Ca⁺⁺ ions levels. These levelsare important for cell viability and function. Thus, intracellular Ca⁺⁺ion concentrations are implicated in a number of vital processes inanimals, such as neurotransmitter release, muscle contraction, pacemakeractivity, and secretion of hormones.

[0008] It is believed that calcium channels are relevant in certaindisease states. A number of compounds useful in treating variouscardiovascular diseases in animals, including humans, are thought toexert their beneficial effects by modulating functions ofvoltage-dependent calcium channels present in cardiac and/or vascularsmooth muscle. Many of these compounds bind to calcium channels andblock, or reduce the rate of, influx of Ca⁺⁺ ions into the cells inresponse to depolarization of the cell membrane. An understanding of thepharmacology of compounds that interact with calcium channels in otherorgan systems, such as the central nervous system, and the ability torationally design compounds that will interact with these specificsubtypes of human calcium channels to have desired therapeutic, e.g.,treatment of neurodegenerative disorders, effects have been hampered byan inability to independently determine how many different types ofcalcium channels exist or the molecular nature of individual subtypes,particularly in the CNS, and the unavailability of pure preparations ofspecific channel subtypes, i.e., systems to evaluate the specificity ofcalcium channel-effecting compounds.

[0009] Multiple types of calcium channels have been detected based onelectrophysiological and pharmacological studies of various mammaliancells from various tissues (e.g., skeletal muscle, cardiac muscle, lung,smooth muscle and brain) Bean, B. P., Annu Rev. Physiol. 51:367-384(1989) and Hess, P., Annu.Rev. Neurosci. 56:337 (1990). These differenttypes of calcium channels have been broadly categorized into fourclasses, L-, T-, N-, and P-type, distinguished by current kinetics,holding potential sensitivity and sensitivity to calcium channelagonists and antagonists. Four subtypes of neuronal voltage-dependentcalcium channels have been proposed Swandulla, D. et al., TrendsNeurosci 14:46 (1991). The L-, N- and P-type channels have each beenimplicated in nociception, but only the N-type channel has beenconsistently implicated in acute, persistent and neuropathic pain. Asynthetic version of ω-conotoxin MVIIA, a 25-amino acid peptide derivedfrom the venom of the piscivorous marine snail, Conus magus has beenused intrathecally in humans and has 85% success rate for the treatmentof pain with a greater potency than morphine.

[0010] While known drug therapies have utility, there are drawbacks totheir use. For instance, it may take up to six months of consistent useof some medications in order for the product to have effect in relievingthe patient's pain. Consequently, a particular subject may be receivingtreatment and continuing to suffer for up to six months before thephysician can assess whether the treatment is effective. Many existingdrugs also have substantial adverse side effects in certain patients,and subjects must therefore be carefully monitored. Additionally, mostexisting drugs bring only temporary relief to sufferers and must betaken consistently on a daily or weekly basis for continued relief.Finally, with disease progression, the amount of medication needed toalleviate the pain may increase thus increasing the potential for sideeffects. Thus, there is still a need for an effective and safe treatmentto alleviate pain.

SUMMARY OF THE INVENTION

[0011] In one aspect the present invention provides compounds havingselective action at N-type calcium channels that are useful for thetreatment of pain.

[0012] Compounds of the present invention that show selective action atN-type calcium channels are compounds in accord with structural diagramI,

[0013] wherein:

[0014] R¹ is halophenyl;

[0015] R² is NE¹E² where E¹ is selected from hydrogen and C₁₋₃alkyl andE² is selected from C₁₋₃alkyl and (CH₂)_(n)phenyl where n is selectedfrom 1, 2 or 3, and

[0016] R³ is selected from phenyl, 1,3-benzodioxolyl and phenylsubstituted with one, two or three moieties independently selected fromhalo, C₁₋₃alkyl, perhaloC₁₋₃akyl, HC(O)—, C₁₋₃alkoxy andC₁₋₃alkylcarbonyl.

[0017] Particular compounds of the inventions are those wherein:

[0018] R¹ is fluorophenyl;

[0019] R² is NE¹E² where E¹ is selected from hydrogen and methyl, and E²is methyl, and

[0020] R³ is selected from phenyl, 1,3-benzodioxol-5-yl and phenylsubstituted with one, two or three moieties independently selected fromchloro, fluoro, methyl, ethyl, methoxy, ethoxy, trifluoromethyll,HC(O)—, and CH₃C(O)—.

[0021] More particular compounds of the invention are those wherein:

[0022] Particular compounds of the inventions are those wherein:

[0023] R¹ is 3-fluorophenyl;

[0024] R² is NE¹E² where E¹ is selected from hydrogen and methyl, and Eis methyl, and

[0025] R³ is selected from phenyl, 1,3-benzodioxol-5-yl and phenylsubstituted with one, two or three moieties independently selected fromchloro, fluoro, methyl, ethyl, methoxy, ethoxy, trifluoromethyll,HC(O)—, and CH₃C(O)—.

[0026] Most particular compounds of the invention are those exemplifiedherein.

[0027] In another aspect, the invention comprises a method for usingcompounds according to structural diagram I for the treatment of pain,said method comprising administering a pain-ameliorating effectiveamount of any such compound.

[0028] One embodiment of the method of the invention comprisesadministering a pain-ameliorating effective amount of a compound inaccordance with structural diagram I to a subject in need of treatmentfor acute, persistent or neuropathic pain.

[0029] In a further aspect, the invention comprises methods for makingcompounds in accord with structural diagram I.

[0030] In yet another aspect, the invention comprises pharmaceuticalcompositions comprising compounds in accord with structural diagram Itogether with excipients, diluents or stabilisers, as further disclosedherein, useful for the treatment of acute, persistent and neuropathicpain.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Compounds of the invention are those within the scope of thegeneric description and particularly those compounds exemplifiedhereafter.

[0032] Suitable pharmaceutically-acceptable salts of compounds of theinvention include acid addition salts such as methanesulphonate, karate,hydrochloride, hydrobromide, citrate, tris(hydroxymethyl)aminomethane,maleate and salts formed with phosphoric and sulphuric acid.

[0033] Where compounds of the present invention possess a chiral centerit is to be understood that the invention encompasses all opticalisomers and diastereoisomers of such compounds.

[0034] Where compounds of the present invention can tautomerize it is tobe understood that the invention encompasses all tautomeric forms ofsuch compounds.

[0035] Where compounds of the present invention can exist in unsolvatedas well as solvated forms such as, for example, hydrated forms, it is tobe understood that the invention encompasses all such solvated andunsolvated forms.

[0036] Another aspect of the invention provides processes for makingcompounds of the invention, as follows:

[0037] a) reacting a substituted acetophenone according to structuraldiagram II with acetic anhydride and sodium hydride to form a3-oxo-propionic acid ethyl ester according to structural diagram III, asfollows:

[0038] b) reacting a compound of structural diagram III with4-bromoaniline to form a 3-substituted 3-(4-bromophenylamino)acrylicacid butyl ester according to structural diagram IV, as follows:

[0039] c) cyclizing a compound of structural diagram IV to form a2-substituted 6-bromo-4-hydroxy-quinoline according to structuraldiagram V, as follows:

[0040] d) chlorinating a compound of structural diagram V to form acompound according to structural diagram VI, as follows:

[0041] e) selectively replacing the chlorine moiety of a compound ofstructural diagram VI to form a compound according to structural diagramVII, as follows:

[0042] f) selectively replacing the bromine moiety of a compound ofstructural diagram VII by reaction with a substituted boronic acid toform a compound according to structural diagram I, as follows:

[0043] wherein, if necessary, in steps a), b), c), d), e) and f) anyfunctional group is protected with a protecting group, and thereafter,

[0044] g) removing any said protecting group;

[0045] h) converting one compound according to structural diagram I toanother compound according to structural diagram I by proceduresdescribed in Methods A through L herein, and

[0046] i) purifying said compound of structural diagram I to the extentnecessary and, if necessary, forming a pharmaceutically-acceptable salt.

[0047] To use a compound of the invention or apharmaceutically-acceptable salt thereof for the therapeutic treatment,which may include prophylactic treatment, of pain in mammals, which maybe humans, the compound can be formulated in accordance with standardpharmaceutical practice as a pharmaceutical composition. Accordingly, afurther aspect of the invention provides a pharmaceutical compositionwhich contains a compound of the structural diagram I as defined hereinor a pharmaceutically-acceptable salt thereof, in association with apharmaceutically-acceptable additive such as an excipient or carrier.

[0048] Suitable pharmaceutical compositions that contain a compound ofthe invention may be administered in conventional ways, for example byoral, topical, parenteral, buccal, nasal, vaginal or rectaladministration, or by inhalation. For these purposes a compound of theinvention may be formulated by means known in the art in the form of,for example, tablets, capsules, aqueous or oily solutions, suspensions,emulsions, creams, ointments, gels, nasal sprays, suppositories, finelydivided powders or aerosols for inhalation, and for parenteral use(including intravenous, intramuscular or infusion) sterile aqueous oroily solutions or suspensions or sterile emulsions. A preferred route ofadministration is orally by tablet or capsule.

[0049] In addition to a compound of the present invention, apharmaceutical composition of this invention may also contain one ormore other pharmacologically-active agents. Alternatively, apharmaceutical composition comprising a compound of this invention maybe co-administered simultaneously or sequentially with one or more othercompatible pharmacologically-active agents.

[0050] Pharmaceutical compositions of this invention will normally beadministered so that a pain-ameliorating effective daily dose isreceived by the subject. The daily dose may be given in divided doses asnecessary, the precise amount of the compound received and the route ofadministration depending on the weight, age and sex of the patient beingtreated and on the particular disease condition being treated accordingto principles known in the art. A preferred dosage regime is once daily.

[0051] A yet further embodiment of the invention provide the use of acompound of the structural diagram I, or a pharmaceutically-acceptablesalt thereof, in the manufacture of a medicament useful for binding toN-type calcium channels in a warm-blooded animal such as a human being.

[0052] Still another embodiment of the invention provides a method ofbinding a compound of the invention to N-type calcium channels of awarm-blooded animal, such as a human being, in need of treatment forpain, which method comprises administering to said animal an effectiveamount of a compound of structural diagram I or apharmaceutically-acceptable salt thereof.

[0053] A further aspect of the present invention provides apharmaceutical composition which includes a compound of the presentinvention as defined herein or a pharmaceutically-acceptable saltthereof, in association with a pharmaceutically-acceptable additive suchas an excipient or a carrier.

[0054] A still further aspect of the present invention is a method oftreatment of the human or animal body that includes the administrationof a compound of the present invention or a pharmaceutically-acceptablesalt thereof.

[0055] Definitions:

[0056] When used herein “halo” or “halogen” means fluoro, chloro, bromoor iodo;

[0057] when substituents herein are stated to be “selected from” or“independently selected from” a group of moieties, it is to beunderstood that included compounds are those where all substituents arethe same and compounds where each substituent is different;

[0058] when used herein the term “alkyl,” as in for example C₁₋₆allyl,unless otherwise defined, includes both straight and branched chainalkyl groups. References to individual alkyl groups such as “propyl”mean the normal, straight chain form, that is, n-propyl;

[0059] when used herein, a term such as “C₁₋₆alkyl” means alkyl groupshaving 1, 2, 3, 4, 5 or 6 carbon atoms and collective groups such asC₁₋₄alkyl and includes straight and branched moieties such as methyl,ethyl, iso-propyl and t-butyl, similarly, a term such as “C₁₋₃alkoxy”includes particular moieties such as methoxy, ethoxy and propoxy, andterms used herein that are not otherwise defined are intended to havetheir conventionally-understood meaning.

[0060] The Methods and Examples which follow are intended to illustratebut not limit the invention. In the Methods and Examples, unlessotherwise stated:

[0061] concentrations were carried out by rotary evaporation in vacuo;

[0062] operations were carried out at ambient temperature, that is inthe range 18-26° C. and under a nitrogen atmosphere;

[0063] column chromatography (by the flash procedure) was performed onMerck Kieselgel silica (Art. 9385);

[0064] yields are given for illustrative purposes only and are notnecessarily the maximum attainable;

[0065] the structure of compounds of the invention were generallyconfirmed by conventional NMR and mass spectral techniques, peakmultiplicities are shown thus: s, singlet; bs, broad singlet; d,doublet; AB or dd, doublet of doublets; t, triplet; dt, double oftriplets; m, multiplet; bm, broad multiplet; FAB m/s data were obtainedusing a Platform spectrometer (supplied by Micromass) run inelectrospray and, where appropriate, either positive ion data ornegative ion data were collected, herein (M+H)⁺ is provided;

[0066] purity of intermediates were was in general assessed by m/s orNMR analysis; and where used the following abbreviations have meaningsas follows: DCM is dichloromethane, DMF is N,N-dimethylformamide, DMSOis dimethylsulfoxide, CDCl₃ is deuterated chloroform, FAB is fast atombombardment, m/s is mass spectroscopy or mass spectral, NMR is NuclearMagnetic Resonance, NMP is N-methylpyrrolidinone, and THF istetrahydrofuran.

[0067] Biological Methods:

[0068] I. N-Channel FLIPR (Fluorescent Laser Imaging Plate Reader)Assay.

[0069] The methods described herein provide a reliable FLIPR-basedreadout of the efficacy and potency with which test compounds inhibitcalcium flux through the N-type calcium channel expressed in its nativeform in a human-derived neuroblastoma cell line differentiatedchemically to a neuronal phenotype. The degree to which a compound at aparticular concentration inhibited the N-channel calcium flux wasdetermined by comparing the amplitude of peak calcium increase in thepresence of the compound to a control 80 mM K⁺ stimulus in wells withoutcompound. Results obtained for this FLIPR assay were validated in twoways:

[0070] a) the N-channel specific peptide toxin, conotoxin MVIIA, showedan IC₅₀=3 nM (determined from fit to five-point concentration responseanalysis), compatible with the known literature value; and

[0071] b) IC₅₀ values were determined for a set of 18 small moleculesfrom chemistry lead series (pIC₅₀ range: 4.67-7.02).

[0072] Potency of these same test compounds as inhibitors of the N-typecalcium current was also determined by direct electrophysiologicalmeasurement either in neuronally differentiated IMR-32 cells, or infreshly-isolated rat superior cervical ganglion neurons. pIC₅₀'s yieldedby the two methodologies for the compound set were closely comparable(r=0.91; p<0.001).

[0073] A. Cell Culture.

[0074] An immortalized cell line, IMR32, derived from humanneuroblastoma cells obtained from the ATCC (product #CCL-127) was usedfor all experiments. Cells were grown in T75 flasks containing Eagle'sminimum essential medium (MEM) w/Earle's salts and non-essential aminoacids without glutamine (Cat. #SLM-034B, Specialty Media, Philipsburg,N.J.), 10% FBS and 1% glutamine. Cells were grown to ˜70-80% confluency(by visual microscopic estimation) before sub-culturing. To maintain astock culture, cultures were split at a ratio of 1:3-1:4 by creating acell suspension by trituration, and pipetting a volume of the cellsuspension sufficient to yield this final ratio into new flaskscontaining ˜20 mL of fresh media. Sub-culturing was generally performedtwo times per week. For preparation of 96 well plates (black-walled; Cat#3603, Costar Co., Cambridge, Mass.), a T75 flask containing cells ofdesired confluency was brought up to 120 ml volume with media. Cellswere then freed by trituration, and the cell suspension was plated into12-96 well plates to yield final well volume of 100 μL.

[0075] B. Cell Differentiation to Neuronal Phenotype.

[0076] Cells were induced to differentiate in a differentiation mediumconsisting of: MEM, 10% FBS, 1% glutamine, 1 μM 2-butyl-cAMP (49.1mg/100 mL media (Cat. #D-0627, Sigma Corp., St Louis, Mo.), and 2.5 mMbromo-deoxy-uridine (stock: 30.7 mg/10 mL media, 25 mL of abovestock/100 mL media; Sigma Cat. #B-9285). To induce differentiation, thecells were treated with differentiation media (by complete mediumchange) 2 days after an initial plating in 96 well plates. Confluency atthis time was ˜40%. A complete medium change with freshly prepareddifferentiating medium was subsequently performed every 2-3 days. Cellswere exposed to these differentiation conditions for 6 to 11 days beforebeing used in FLIPR experiments.

[0077] C. Standard Experimental Solutions.

[0078] Solutions of the following composition (in mM) were used inexperiments (Buffers without probenicid purchased from Specialty Media(Buffers A and B: Cat. #BSS053A; Buffers C & D: Cat. #BSS056A).

[0079] Buffer A (first wash buffer): Krebs-Ringer-HEPES (KRH) buffer:NaCl: 125, KCl: 5, MgSO₄: 1.2, KH₂PO₄: 1.2, CaCl₂ 2H₂O: 2, Glucose: 6,HEPES: 25, pH: 7.4 (pH adjusted with NaOH)

[0080] Buffer B (dye loading buffer) KRH buffer with 2.5 μM probenicid:same as buffer A, but probenicid added to final concentration of 2.5 μM.Probenecid (Cat. #P-8761, Sigma Chemical Co., St. Louis, Mo.) made as astock solution at 250 mM.

[0081] Buffer C (dye washout buffer) KRH buffer with 0 mM K⁺ and 2.5 μMprobenicid: NaCl: 130, MgSO₄:1.2, NaH₂PO₄: 1.2, CaCl₂2H₂O: 2, Glucose:6, HEPES: 25, pH; 7.4 (pH adjusted with NaOH).

[0082] Buffer D (compound dilution buffer): Buffer C with 0.1% w/vbovine serum albumin (BSA; Sigma).

[0083] D. Pharmacological Standards and Compounds.

[0084] The following solutions were used to obtain the data disclosedherein.

[0085] Nitrendipine: (RBI Chemicals, Natick, Mass.): Stock: 10 mM inDMSO; Pipetting solution: 9 μM; pipette 20 μL into 120 μL volume in wellfor final well concentration: 1 μM.

[0086] w-Conotoxin MVIIA: (Cat. #H-8210; Bachem Inc., Torrance, Calif.):Stock: 1 mM in HPLC grade H₂O with 0.1% BSA; Pipetting solution: 4.5 μM;pipette 20 μl into 140 μl volume in well for final well concentration: 1μM.

[0087] Test compound stock and solution preparation: Compounds prepareddaily as stocks at 10 mM in 100% DMSO; Pipetting solution: 45 μM orserial dilutions thereof; pipette 20 μL into 140 μL volume in well forfinal well concentration: 1 μM or 10-fold dilutions thereof.

[0088] High potassium (depolarization) solution: Buffer C with 240 mM K⁺added; pipette 80 μL into 160 μL volume in well for final wellconcentration of 80 mM K⁺.

[0089] E. Cell Loading with Fluorescent Dyes.

[0090] Fluorescent dye solution preparation: A calcium indicator dye,Fluo-4 acetylmethylester (Fluo 4-AM; Cat. #F-124201; Molecular Probes,Eugene, Oreg.) was used to measure changes in intracellular free calciumwith FLIPR. 1 mM Fluo-4 AM stock solution was made by dissolution inDMSO. This stock was then diluted to 4.6 μM with Buffer B (Fluo-4 AMworking solution).

[0091] Cell loading procedure: Plates containing cells were washed withBuffer A using an automated cell washer (Model #: 5161552, LabsystemsOy, Helsinki, Finland) with controls set to the following parameters:cell height: C/D; cell pulse: 4/5, washes: 3; volume: 5; DRY positionsetting. These settings resulted in a 70 μL residual depth of bufferover cells in each well. 100 μL of the Fluo-4 AM working solution wasthen added to each well resulting in a final Fluo-4 AM concentration of2.7 μM Cells were incubated in this solution at 37° C. for 1-1.5 h.Cells were then washed with Buffer C five times using the cell washerwith parameters the same as the pre-loading washes above with theexceptions of: washes: 5; WET position setting. A final wash was thenconducted by changing the parameters as follows: washes: 1; volume: 2.This resulted in a final well volume of 120 μL. Cells were allowed toequilibrate under this condition for 10 min, and then used in the FLIPRprotocol.

[0092] F. FLIPR Protocol

[0093] Instrumentation: Real time changes in intracellular free calciumin response to potassium-induced depolarization in the absence orpresence of putative N-channel inhibitors were measured by either aFLIPR I or FLIPR II (configured for 96-well format) instrument(Molecular Devices, Sunnyvale, Calif.). Identical settings and protocolswere used with each instrument, and results obtained from the twoinstruments were indistinguishable for a set of standard benchmarkcompounds.

[0094] FLIPR hardware settings: Laser power was set to about 0.3 watts.Excitation wavelength was set to a 488 nm peak, and the emissionwavelength to 540 nm. Camera aperture was set to 2. All experiments wereconducted at room temperature (20-22° C.).

[0095] Plate layout—reference signals: Certain wells on each plate wereallocated to standards to determine minimum and maximum specificfluorescent signal against which inhibitory effects of compounds werenormalized. The reference standards were distributed at plate locationsincluding edge and interior wells

[0096] Maximum signal (N-channel+non-specific): 12 wells were incubatedin nitrendipine (1 μM) solution and 80 mM K⁺added to determine maximalCa²⁺ increase mediated by N-channels+non-specific (non-L, non-N-channelmediated fluorescence increase). The coefficient of variation amongstthese wells for the K⁺-evoked peak increase in fluorescence units wastypically less than 12%.

[0097] Minimum signal (non-specific): 6 wells were incubated innitrendipine (1 μM)+w-conotoxin MVIIA and 80 mM K⁺ added to determinebackground Ca²⁺ with all N-channels pharmacologically occluded. The peaknon-specific signal component was typically less than 15% of the maximumsignal peak amplitude.

[0098] N-channel reference small molecule: A compound that had beencharacterized extensively with respect to N-channel inhibitory activityin both FLIPR and patch clamp electrophysiology was included on eachplate in triplicate at 1 μM (near IC₅₀) to establish a reference point.

[0099] Test compounds: 5 test compounds were evaluated for potency oneach plate. Each compound was tested at 5 increasing concentrationsspanning half-log units and typically reaching a maximal concentrationof 10 μM. Each concentration was tested in triplicate wells.

[0100] Protocol structure: The FLIPR protocol was configured as threesolution addition/sampling sequences (see below). Conotoxin (1 μM finalconc.) was added to appropriate wells prior to placing the plate in theFLIPR instrument. Wells initially contained a total solution volume of100 μl, and after all three solution additions contained 240 μl. Theactive mixing (by the pipette) option was not used in any sequence.

[0101] Nitrendipine addition sequence: 28 s total duration withfluorescence signal sampling at 1 Hz for 2 s, followed by addition of 20μL nitrendipine standard solution at 10 μL/s, followed by sampling at0.5 Hz for 24 s.

[0102] Test compound addition sequence: 64 s total duration withsampling at 0.5 Hz for 4 sec, test solution addition of 40 μL at 20μL/s, followed by sampling at 0.2 Hz for 60 s.

[0103] Compound incubation, cell depolarization and calcium readoutsequence: 1024 s total duration with sampling at 0.0167 Hz for 840 s,followed by solution addition 80 μL of high K⁺ (depolarization)solution, followed by sampling at 1 Hz for 180 sec. This final 180 secsampling interval thus represented the epoch where the peak increase inintracellular calcium due to flux through activated N-channels occurred.

[0104] G. Data Analysis

[0105] FLIPR software: Prior to export, the data was normalized withinthe FLIPR software module for two effects.

[0106] Baseline correction: The baseline was corrected by “zeroing” atsample #57 (immediately prior to KCl addition). This normalizationserved to correct the y axis offset of the fluorescent trace from eachwell so that all traces had a common point just prior to onset of therelevant evoked fluorescent increase.

[0107] Spatial uniformity correction factor: The data was normalized bya procedure which calculates a mean over the plate of fluorescent unitsfrom the first sample, and then multiplies the data from each well by ascalar that adjusts the value of the first sample to this average value,thus normalizing for differences in absolute baseline fluorescenceamongst the wells caused by differences in cell densities or dyeloading.

[0108] External software: Data were exported from FLIPR into Excel as“*.squ” extension files. Following export, operations were performed inExcel to calculate the maximal peak amplitude (relative to the zeroedbaseline) of the fluorescence increase following potassium addition ineach well. Measurements from wells where an test compound was added werethen normalized as a percentage between the mean amplitudes from thereference wells providing the maximum (100%) and non-specific (0%)signal components, as described above. The resulting percent inhibitionby test compounds was considered to reflect inhibition of calcium fluxat the N-type channel.

[0109] II. L-Channel FLIPR Assay.

[0110] The methods described below provided a reliable FLIPR-basedreadout of the efficacy and potency with which test compounds inhibitedcalcium flux through the L-type calcium channel expressed natively in ahuman-derived neuroblastoma cell line, SK—N—SH. The degree to which agiven compound concentration inhibited the L-channel was determined bycomparing the amplitude of peak calcium increase to an 80 mM K⁺ stimulusin the test well to the peak increase in wells without compound Theassay was validated by obtaining 5-point concentration-response curvesand thereby determining IC₅₀ values for the reference L-channelblockers, nitrendipine (30 nM), nifedipine and verapamil. These valueswere compatible with the known literature values for these agents toblock Ca²⁺ flux through the L-channel,

[0111] A. Cell Culture:

[0112] An immortalized cell line, SK—N—SH, derived from humanneuroblastoma cells (ATCC product #HTB-11) was used for all experiments.Cells were grown in T75 flasks containing Eagle's minimum essentialmedium (MEM) w/Earle's salts, with 0.1 mM non-essential amino acids, 1.0mM Na pyruvate and 10% fetal bovine serum (FBS; Cat. #SLM-034B,Specialty Media). Cells were grown to 100% confluency (by visualmicroscopic estimation) before sub-culture. Cells were sub-cultured at aratio of 1:3 by first rinsing with 3 mL PBS, replacing the PBS with PBScontaining 0.25% trypsin until the cells detached from the surface. 1 mLof the resulting suspension was then added to a new flask containing 10mL fresh media. Cells were then incubated (37° C., 5% CO₂), and mediawas exchanged about 3 days after subculturing.

[0113] B. Preparation of Cells for Experiments:

[0114] Cells used for experiments were at the 100% confluency growthstage. Each flask provided enough cells for three 96-well plates. Cellswere detached from the flask by addition of 0.25% trypsin, as describedfor the sub-culturing protocol. Once detached, 7 mL fresh media wasadded to the flask, and the solution triturated gently. An additional 20mL media was then added, and 100 μL of this final cell suspension wasthen added to each well of a 96-well plate. Before use in experimentsthe plates were incubated at 37° C. in 5% CO₂ until cells reached 100%confluence (1-2 days).

[0115] C. Experimental Procedures:

[0116] The composition of solutions, hardware settings, plate layout,structure of the FLIPR protocol, and analytical settings and procedureswere identical to those described herein for the N-channel assays withthe following differences as regards Plate layout and reference signals.

[0117] Maximum signal (L-channel+non-specific): 12 wells received 20 μLbuffer addition only (no nitrendipine) in the first solution additionsequence to define the maximal K⁺-evoked Ca²⁺ increase mediated byL-channels+non-specific (non-L-channel mediated fluorescence increase).The coefficient of variation amongst these wells for the K⁺-evoked peakincrease in fluorescence units was typically less than 12%.

[0118] Minimum signal (non-specific): 6 wells were incubated innitrendipine (1 μM), followed by 80 mM K⁺ added to determine backgroundCa²⁺ with all L-channels pharmacologically occluded. The peaknon-specific signal component was typically less than 15% of the maximumsignal peak amplitude.

[0119] L-channel reference small molecule: Nitrendipine was included intriplicate wells on each plate at 30 nM (near IC₅₀) for a referencereadout

[0120] III. N-Channel Patch Clamp Electrophysiology.

[0121] Conventional whole cell recording techniques were used todirectly measure the ability of test compounds to inhibit Ca²⁺ currentthrough N-type calcium channels. N-type current were recorded from bothneuronally differentiated IMR-32 cells, and native neurons freshlydissociated from superior cervical ganglia of early postnatal rats. Eachday, currents in both cell types were confirmed as N-currents showingthat greater than 90% of the total inward current during depolarizingsteps was blocked by a supramaximal concentration (3 mM) of w-conotoxinMVIIA. Additionally, the potency of w-conotoxin MVIIA was periodicallydetermined to be about 3 nM (IC₅₀), a value consistent with thatreported in the literature. Results for a subset of compounds tested inboth cell types did not differ significantly, thus data are consideredas one data set unless otherwise specified.

[0122] A. IMR-32 Cell Culture and Differentiation:

[0123] IMR32 cells were cultured and neuronally differentiated usingprocedures identical to those described for the FLIPR N-channel assayexcept that for differentiation cells were plated in 35 mm plexiglassculture dishes, rather than 96-well plates.

[0124] B. Dissociation of Rat Superior Cervical Ganglion (SCG) Neurons:

[0125] 7-10 day old rat pups were euthanized in a chamber containing ahigh CO₂ atmosphere. Immediately, SCG were surgically isolated, removedand placed in ice cold Hanks balance salt solution (HBSS). SCG's weredesheathed, cut open and placed in a solution of HBSS containing 20 U/mLpapain (37° C.) for 15 min. The papain solution was then exchanged forHBSS (37° C.) containing 16 mg/mL dispase and 400 U/mL collagenase for40 min with gentle trituration of tissue every 15 min. Cells were thenrecovered by centrifugation and stored in 115 medium at 4° C. for use onthe same day. For recording, a drop of cell containing solution wasplaced on a poly-L-lysine coated 35 mm plexiglass culture dish, andcells allowed to adhere for several minutes.

[0126] C. Electrophysiological Procedures:

[0127] Solutions: Recording solutions were adapted from those describedby Thompson and Wong (1991.) J. Physiol., 439: 671-689. Solutions werestored as aliquots for not more than one month (intracellular, −20° C.,extracellular, 4° C.) before experiments. The pipette (intracellular)solution contained (in mM): TRIS, 130; CsBAPTA, 10; HEPES, 10; Mg²⁺ ATP,5; pH to 7.3 with methanesulphonic acid; osmolality ˜315 mOsm.Extracellular solution contained (in mM): TRIS 120; CsCl, 5; HEPES, 10;Mg²⁺Cl, 1; Ba²⁺Cl, 5, glucose, 25; tetraethylammonium chloride, 15;tetrodotoxin, 200 (added at time of experiment); pH to 7.4 withmethanesulphonic acid; osmolality ˜320 mOsm.

[0128] Whole cell recording and analysis: The whole-cell voltage clampconfiguration of the patch clamp technique as described by Hamill et al.(1981) Pflügers Arch. 391: 85-100, was employed to isolatevoltage-dependent calcium currents. Culture dishes containing cells wereplaced in a chamber on the stage of an inverted microscope. Allexperiments were conducted at room temperature (20-22° C.). Patchpipettes were fabricated from thin-wall glass (1.5 mm OD, 1.12 mm ID;World Precision Instruments, New Haven, Conn.) on the Brown-Flaming P-86puller (DC resistance: 3-6 MΩ; Sutter Instr. Co., Novato, Calif.). AnAxopatch 1B amplifier (Axon Instruments, Foster City, Calif.) was usedto obtain current signals and this was connected to a personal computerby either a TL-1 (Scientific Solutions, Solon, Ohio) or Digidata 1200(Axon Instr.) interface. The current signal was balanced to zero withthe pipette immersed in the bath just prior to forming a seal on theneuron. Seal resistance ranged from 1 to greater than 10 GΩ. Seriesresistance was usually less than 10 MΩ, and was not compensatedelectronically. Digitized data acquisition and voltage step protocolswere accomplished with pClamp 6.0 software (Axon Instr). Data werelow-pass filtered at less than one-half the digital sampling rate priorto digitizing. To record N-type currents for evaluation of inhibitorypotency of compounds (steady-state concentration-response analysis), 200ms voltage steps to +10 mV were delivered at 15 sec intervals from aholding potential of −90 mV. The recorded currents were leak subtractedon-line with a P-4 or P-6 subpulse protocol in the pClamp software. Toevaluate open channel block of compounds, 10 ms voltage steps to +10 mVwere delivered at varying frequencies from a holding potential of −90 mVwithout using on-line leak subtraction. These voltage protocols bothyielded constant inward current amplitudes over 5-10 minutes ofrecording. Peak current amplitude was analyzed using the clampfit moduleof pClamp software. Origin 5.0 software (Microcal Corp, Northampton,Mass.) was used to iteratively fit concentration-response data to astandard Hill function, and to provide graphic displays for currenttraces and analyzed data.

[0129] Drug/compound preparation and delivery: Test compounds wereprepared as 10 mM stock solutions in DMSO, and appropriate volumes ofthese stock solutions dissolved into extracellular buffer to yield thedesired concentrations. Solutions containing drugs/compounds wereapplied focally from any of six linearly arranged glass-lined tubes (200mm o.d., Hewlett Packard, Wilmington, Del.) positioned ˜100 mm from therecorded neuron. Each solution was released from the desired tube by anelectronically controlled solenoid valve system (BME Systems, Baltimore,Md.). This system achieved rapid (<100 ms) equilibration of drugsolution in the extracellular phase without perturbing the recordingcharacteristics.

[0130] IV. Formalin Test.

[0131] The Formalin test is a well established pain test (Dubuisson andDennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al., 1993) whichassesses the inhibitory effects of orally administered N-type calciumchannel antagonists on formalin-induced nocifensive behaviours in rats.This test consists of two distinct phases of formalin-induced behaviour.The first phase response, occurring between 0 to 5 minutes, is caused byacute nociception to the noxious chemical (formalin) injected into thepaw. This is followed by a quiescent period of between 5 to 15 min postinjection. A second phase response, occurring after 15 minutes andlasting up to 60 minutes, is caused by sensitisation of the centralneurons in the dorsal horn. Central sensitisation augments the noxiousafferent input and a stronger pain barrage is transmitted into thebrain. Inhibition of the second phase response is indicative of acentral mechanism of drug action.

[0132] The procedure for the formalin test is as follows: male rats areplaced in a plexiglass chamber and observed for 30-45 min. to observetheir baseline activity. Multiple groups of animals are pretreated witheither vehicle or different doses of a test compound. Animals are dosedwith the drug of interest either 40 min., if by the intraperitonealroute, or 90 min., if by the oral route, prior to injection of formalininto a hind paw (under the dorsal skin; 0.05 mL of sterile 5% formalin).The number of paw flinches and licks during first phase (0-5 min.) andsecond phase (20-35 min.) are scored and recorded. Flinch and lickresponses are calculated as percentage of inhibition compared with themean score of a saline control group. Drug potencies are expressed asthe dose which causes 50% of the maximum inhibitory effect (“ID₅₀”).Student t-tests are used for statistical analysis to determine thesignificance of drug effects. Compounds are considered active based ontheir ability to inhibit the flinch response.

[0133] V. Chronic Constrictive Injury Test.

[0134] The Chronic Constrictive Injury (“CCI”) test or Neuropathic PainModel assesses neuropathic pain associated with nerve injuries that canarise directly from trauma and compression, or indirectly from diseasesranging from infection to cancer, metabolic conditions, toxins,nutritional deficiencies, immunological dysfunction and musculoskeletalchanges. In the CCI model (Bennett and Xie, 1988) a unilateralperipheral neuropathy is produced in rats by partial nerve ligation.

[0135] Sprague-Dawley rats (250-350 g) are anesthetized with sodiumpentobarbital and the common sciatic nerve is exposed at the level ofthe mid thigh by blunt dissection through the biceps femoris. A sectionof nerve (about 7 mm), proximal to the sciatic trifurcation, is exposedand ligated 4 times with chromic gut suture. The suture is tied withabout 1 mm spacing between ligatures. The incision is closed in layersand the animals are allowed to recover. Thermal hyperalgesia is measuredusing the paw-withdrawal test (Hargreaves et al, 1988). Nervecompression due to the partial nerve ligation causes'shorter latenciesfor paw withdrawal compared to the latency of paw withdrawal of paws ofnormal or sham operated legs. Animals are habituated on an elevatedglass floor. A radiant heat source is aimed at the mid-plantar hindpaw(sciatic nerve territory) through the glass floor with a 20 secondcut-off used to prevent injury to the skin. Latencies for the withdrawalreflex in both paws are recorded. Response to test compounds areevaluated at different times following oral administration to determineonset and duration of drug effect. Dose response studies are conductedwith multiple groups of CCI rats dosed orally with either vehicle or thetest compound for 5 days. Paw withdrawal latencies are measured each dayprior to the first daily dose. Data analysis is performed by multiplemeans comparison (Dunnett's test) and drug potencies are expressed asthe dose which causes 50% of the maximum efficacy (“EC₅₀”).

[0136] Compounds of the invention generally bind to N-type calciumchannels with IC₅₀'s, measured in the FLIPR assay described herein ofabout 10 μM or less. For example, compounds of Examples 12, 13, 14, 15and 16, respectively exhibit IC₅₀'s of 10.53, 8.60, 8.82, 5.38 and 5.78.

[0137] Exemplary Chemical Method:

[0138] The following method describes the preparation of6-(3,5-Dichlorophenyl)-2-(3-fluorophenyl)-N-methyl-4-quinolinamine, thecompound of Example 17, below. Other compounds exemplified herein wereprepared by procedures analogous to those described heretofore fromsuitable substituted-acetophenone and substituted-boronic acidprecursors.

[0139] 3-(3-Fluorophenyl)-3-oxo-propionic acid ethyl ester:

[0140] Into a three-neck 2 L round-bottom flask equipped with anaddition funnel, nitrogen inlet, magnetic stirrer, heating mantle,thermocouple and condenser, was placed 21.7 g (0.543 moles) of a60%-in-oil dispersion of sodium hydride. To this was added 1 L dryhexane and the resulting suspension was stirred for 15 minutes. Stirringwas halted and the solids were allowed to settle and the clearsupernatant containing the hexane and dissolved oil was then removed viaa cannula. Diethyl carbonate (1 L) was added to the solids and thesuspension was heated to 120° C. To the hot suspension was cautiouslyadded dropwise, over 40 minutes, a solution of 100 g (0.494 moles) ofm-fluoro acetophenone dissolved in 250 mL of diethyl carbonate. Asaddition proceeded a reaction initiated, hydrogen was evolved and thecolor changed to tan. After the acetophenone-derivative addition wascomplete, the reaction was heated for 1 additional hour. The reactionmixture was cooled and was poured into a 2 L separatory funnel. Thediethyl carbonate layer was twice washed with 10% acetic acid solution,dried over MgSO₄ and filtered. The product was purified by vacuumdistillation (bp 114-117° C. at 0.8-0.9 mm Hg) in 91% yield.

[0141] 3-(4-Bromo-phenylamino)-3-(3-fluorophenyl)-acrylic acid butylester:

[0142] Into a 1 liter single-neck round-bottom flask equipped with aSoxhlet extractor apparatus with condenser, magnetic stirrer andnitrogen inlet was placed 50.25 g (0.183 moles) of3-(4-cyclohexyl-phenyl)-3-oxo-propionic acid ethyl ester, 25 g (0.167moles) of 4-bromoaniline, 1.55 g (0.008 moles) 4-bromoanilinehydrochloride salt and 500 mL of dry n-butanol. Into the Soxhlet thimble(33×118 mm) was placed highly activated 4 Å sieves (1.7-2.4 mm beads).These sieves are activated immediately before use under high vacuum withheating (400° C. for 30 min). The mixture was then brought to refluxsuch that the butanol azeotropically removed water, driving theequilibrium reaction, and the water was removed from the butanol by thesieves before being returned to the reaction pot. The reaction wasallowed to continue for 48 hrs. It was necessary to replace the chargeof sieves after the first 24 hrs. Transesterification to the butyl esteralong with removal of ethanol occurs concomitantly with enamineformation. After 48 hrs the reaction pot was cooled, then placed in a−40° C. freezer and crystals were allowed to form over 24 hrs. Thecrystals were collected by vacuum filtration and the solids washed withcold ethanol. The product was then dried in a vacuum oven to give 73.8 g(98%) of the desired enanine.

[0143] 6-Bromo-2-(3-fluorophenyl)-quinolin-4-ol:

[0144] To a 3 L three-neck flask, equipped with mechanical stirrer,Claisen adapter holding a thermocouple probe and reflux condenser withnitrogen inlet was added 0.75 L of Dowtherm A (a eutectic mixture of26.5% diphenyl and 73.5% diphenyl oxide) and the solvent was heated to250° C. To this was cautiously added in small portions 77.0 grams (0.215moles) of 3-(4bromo-phenylamino)-3-(3-fluorophenyl)-acrylic acid butylester over the course of 0.25 hours. The mixture was maintained at 250°C. for 1.5 hours and then allowed to cool to 90° C. over the course of 2hours. The mixture was treated with 1.0 L of hexanes, and allowed tocool to room temperature while stirring overnight. The tan solids werecollected by suction filtration and washed with three 0.15 L portions ofhexanes. The solids were dried under vacuum at 50° C. overnight to yield58.63 grams (96.0%) of the title compound.

[0145] 6-Bromo-4chloro-2-(3-fluorophenyl)-quinoline:

[0146] Into a 500 mL three-neck round-bottom flask equipped with acondenser, magnetic stirrer and nitrogen inlet was placed 5.2 g (16.3mmoles) of 6-bromo-2-(3-fluorophenyl)-quinolin-4-ol. To this was added15.2 mL( 25.0 g, 163 mmoles, 10 equiv.) of phosphorus oxychloride withstirring. The mixture was heated to 110° C. for 4 hr. At the end of thistime the reaction was cooled to room temperature and water wascautiously added dropwise until all of the POCl₃ was consumed. Theproduct crystallised from the water and solids were collected byfiltration. The solids were washed with water, placed in a 250 mLErlenmeyer and triturated with water. After collection by filtration,washing with water, and drying in a vacuum oven, 4.6 g (84%) of theproduct was obtained.

[0147] N-[6-Bromo-2-(3-fluorophenyl)-4-quinolinyl]-N,N-dimethylamine:

[0148] Into a 500 mL three-neck round-bottom flask equipped withmagnetic stirrer, nitrogen inlet, gas outlet, condenser and heating bathwas placed 20 g (59.4 mmoles of6-bromo-4-chloro-2-(3-fluorophenyl)-quinoline. The material wasdissolved in 150 mL of N-methyl pyrrolidinone and 250 mL of a 40% aq.solution of dimethylamine was added to the stirring mixture. Thereaction was then warmed to 60° C. for 48 hrs. At the end of this timethe reaction was cooled, into 3 L of water in a 4 L Erlenmeyer flask andthe mixture was stirred until solids formed. The solids were collectedby vacuum filtration and dried in a vacuum oven. The product wasrecrystallised from ethanol in a −20° C. freezer to give 19.6 g (95%)yield of the aminated product.

[0149] N-[6-bromo-2-(3-fluorophenyl)-4-quinolinyl]-N,N-dimethylamine wasalternatively prepared as follows. Into a 1 L Parr bomb equipped withmechanical stirring, thermocouple, heater with controller and pressuregauge was placed 20 g (59.4 mmoles of6-bromo-4-chloro-2-(3-fluorophenyl)-quinoline. To this was added 350 mLof ethanol and 350 mL of a 40% aq. solution of dimethylamine. The bombwas sealed and the stirred mixture was then heated to 100° C. resultingin a pressure of approximately 15.0 psi. Heating was continued for 24hrs. At the end of this time the reaction was allowed to cool to roomtemperature and was then vented. The contents were then poured into 3 Lof water in a 4 L Erlenmeyer flask and the mixture was stirred untilsolids formed. The solids were collected by vacuum filtration and thendried in a vacuum oven. The crude product was recrystallised fromethanol in a −20° C. freezer to give 18.15 g (92%) yield of the aminatedproduct.

[0150]6-(3.5-Dichlorophenyl)-2-(3-fluorophenyl)N-methyl-4-quinolinamine:

[0151] To 0.4 g. (1.2 mmole) ofN-[6-bromo-2-(3-fluorophenyl)4-quinolinyl]-N-methylamine was added 0.275g. (1.45 mmole) of 3,5-dichlorophenylboronic acid and a catalytic amountof tris(dibenzylideneacetone)dipalladium (0), 0.0109 g. (0.012 mmole).The mixture was dissolved in 7 mL of N-methyl-2-pyrrolidinone. Sodiumcarbonate, 0.105 g. (1.45 mmole) was dissolved in a minimal amount ofwater and added in one portion to the N-methyl-2-pyrrolidinone reactionsolution. The mixture was heated to 80° C. for 10 hours. After coolingto room temperature, the mixture was diluted to 10 mL total volume withmethanol and filtered through a silica plug. The title compound wasisolated by reverse phase HPLC on a 2 inch Dynamax phenyl column using asolvent gradient from 85:15 water to 100 % methanol, yield 0.320 g.(67%).

EXAMPLES

[0152] Exemplary compounds 1 to 22, inclusive, are disclosed in Table 1which shows the name of each compound, the molecular formula and themass spectroscopy result determined for the particular compound. TABLE 1Molecular Ex. Name Formula M + 1 1 2-(3-fluorophenyl)-6-(4- C₂₃H₁₈F₂N₂361(+) fluorophenyl)-4- dimethylaminoquinoline 22-(3-fluorophenyl)-6-(4- C₂₄H₂₁FN₂O 373(+) methoxyphenyl)-4-dimethylaminoquinoline 3 2-(3-fluorophenyl)-6-(1,3- C₂₄H₁₉FN₂O₂ 387(+)benzodioxol-5-yl)- 4-dimethylaminoquinoline 42-(3-fluorophenyl)-6-(3-ethan-1-onyl- C₂₅H₂₁FN₂O 385(+)phenyl)-4-dimethylaminoquinoline 5 2-(3-fluorophenyl)6-(2,4- C₂₃H₁₇F₃N₂379(+) difluorophenyl)--4- dimethylaminoquinoline 62-(3-fluorophenyl)-6-(4- C₂₄H₂₁FN₂ 357(+) methylphenyl)-4-dimethylaminoquinoline 7 2-(3-fluorophenyl)-6-(3,4,5- C₂₆H₂₅FN₂O₃ 433(+)trimethoxyphenyl)-4- dimethylaminoquinline 8 2-(3-fluorophenyl)-6-[3-C₂₄H₁₈F₄N₂ 411(+) (trifluoromethyl)phenyl]-4- dimethylamnioquinoline 92-(3-fluorophenyl)-6-(2- C₂₄H₂₁FN₂ 357(+) methylphenyl)-4-dimethylaminoquinoline 10 2-[4-(dimethylamino)-2-(3- C₂₄H₁₉FN₂O 371(+)fluorophenyl)-6-quinolinyl] benzaldehyde. 11 2-(3-fluorophenyl)-6-(2-C₂₄H₂₁FN₂O 373(+) ethoxyphenyl)-4- methylaminoquinoline 122-(3-fluorophenyl)-6-(2- C₂₂H₁₆ClFN₂ 363(+) chlorophenyl)-4-methylaminoquinoline 13 2-(3-fluorophenyl)-6-phenyl-4- C₂₂H₁₇FN₂ 329(+)methylaminoquinoline 14 2-(3-fluorophenyl)-6-(2- C₂₃H₁₉FN₂ 343(+)methylphenyl)-4- methylaminoquinoline 15 2-[2-(3-fluorophenyl)-4-C₂₃H₁₇FN₂O 357(+) (methylamino)-6-quinolinyl] benzaldehyde 162-(3-fluorophenyl)6-(2- C₂₃H₁₉FN₂O 359(+) methoxyphenyl)-4-methylaminoquinoline 17 2-(3-fluorophenyl)-6-(3,5- C₂₂H₁₅Cl₂FN₂397/399(+) dichlorophenyl)-4- methylaminoquinoline 182-(3-fluorophenyl)-6-(1,3- C₂₃H₁₇FN₂O₂ 373(+) benzodioxol-5-yl)-4-methylaminoquinoline 19 2-(3-fluorophenyl)-6-(4- C₂₂H₁₆F₂N₂ 347(+)fluorophenyl)-4- methylaminoquinoline 202-(3-fluoro-phenyl)-6-(3-chloro-5- C₂₃H₁₈ClFN₂ 377/379(+)methyl-phenyl)-4- methylaminoquinoline 21 2-(3-fluorophenyl)-6-(4-C₂₃H₁₈ClFN₂ 377/379(+) chlorophenyl)-4- methylaminoquinoline 222-phenyl-6-phenyl-4-pheneth-2- C₂₉H₂₄N₂ 401(+) ylaminoquinoline

1. Any compound in accord with structural diagram I,

wherein: R¹ is halophenyl; R² is NE¹E² where E¹ is selected fromhydrogen and C₁₋₃alkyl and E² is selected from C₁₋₃alkyl and(CH₂)_(n)phenyl where n is selected from 1, 2 or 3, and R³ is selectedfrom phenyl, 1,3-benzodioxolyl and phenyl substituted with one, two orthree moieties independently selected from halo, C₁₋₃alkyl,perhaloC₁₋₃alkyl, HC(O)—, C₁₋₃alkoxy and C₁₋₃alkylcarbonyl.
 2. Acompound according to claim 1, wherein: R¹ is fluorophenyl; R² is NE¹E²where E¹ is selected from hydrogen and methyl, and E² is methyl, and R³is selected from phenyl, 1,3-benzodioxol-5-yl and phenyl substitutedwith one, two or three moieties independently selected from chloro,fluoro, methyl, ethyl, methoxy, ethoxy, trifluoromethyll, HC(O)—, andCH₃C(O)—.
 3. A compound according to claim 2, wherein: R¹ is3-fluorophenyl; R² is NE¹E² where E¹ is selected from hydrogen andmethyl, and B² is methyl, and R³ is selected from phenyl,1,3-benzodioxol-5-yl and phenyl substituted with one, two or threemoieties independently selected from chloro, fluoro, methyl, ethyl,methoxy, ethoxy, trifluoromethyll, HC(O)—, and CH₃C(O)—.
 4. A method fortreating pain, said method comprising administering a pain-amelioratingeffective amount of a compound according to claim
 1. 5. A methodaccording to claim 5, comprising administering a pain-amelioratingeffective amount of a compound according to claim 1 to a subject in needof treatment for acute, persistent or neuropathic pain.
 6. A method formaking compounds according to claim 1, said method comprising: a)reacting a substituted acetophenone according to structural diagram IIwith acetic anhydride and sodium hydride to form a 3-oxo-propionic acidethyl ester according to structural diagram III, as follows:

b) reacting a compound of structural diagram III with 4-bromoaniline toform a 3-substituted 3-(4-bromophenylamino)acrylic acid butyl esteraccording to structural diagram IV, as follows:

c) cyclizing a compound of structural diagram IV to form a 2-substituted6-bromo4 hydroxy-quinoline according to structural diagram V, asfollows:

d) chlorinating a compound of structural diagram V to form a compoundaccording to structural diagram VI, as follows:

e) selectively replacing the chlorine moiety of a compound of structuraldiagram VI to form a compound according to structural diagram VII, asfollows:

f) selectively replacing the bromine moiety of a compound of structuraldiagram VII by reaction with a substituted boronic acid to form acompound according to structural diagram I, as follows:

wherein R¹, R² and R³ are as defined in claim 1; wherein, if necessary,in steps a), b), c), d), e) and f) any functional group is protectedwith a protecting group, and thereafter, g) removing any said protectinggroup; h) converting one compound according to structural diagram I toanother compound according to structural diagram I by proceduresdescribed in Methods A through L herein, and i) purifying said compoundof structural diagram I to the extent necessary and, if necessary,forming a pharmaceutically-acceptable salt.
 7. A pharmaceuticalcomposition for the treatment of acute, persistent or neuropathic paincomprising a compound according to claim 1 together with one or moreadditives selected from excipients, diluents or stabilisers.